Novel human kinase and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/254,744 which was filed on Dec. 11, 2000 and isherein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding a proteinthat shares sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes, which can be used for diagnosis, drug screening,clinical trial monitoring, the treatment of diseases and disorders, andcosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

[0003] Kinases mediate phosphorylation of a wide variety of proteins andcompounds in the cell. Along with phosphatases, kinases are involved ina range of regulatory pathways. Given their physiological importance,kinases have been subject to intense scrutiny and are proven drugtargets.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode a novel human protein,and the corresponding amino acid sequence of this protein. The novelhuman protein (NHP) described for the first time herein sharesstructural similarity with animal kinases, including, but not limitedto, serine/threonine kinases, tyrosine kinases, TGF-beta activatedkinases, and a variety of growth factor receptors. As such, the novelpolynucleotides encode a new kinase protein having homologues andorthologs across a range of phyla and species.

[0005] The novel human polynucleotides described herein, encode an openreading frame (ORF) encoding a protein of 1036 amino acids in length(see SEQ ID NO: 2).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHPs, including small molecules, large molecules, mutant NHPs,or portions thereof, that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHPs. When the unique NHPsequences described in SEQ ID NOS:1-3 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHP sequences described in SEQ IDNOS:1-3 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins that would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses.

[0007] Additionally, the unique NHP sequences described in SEQ IDNOS:1-3 are useful for the identification of protein coding sequence andmapping a unique gene to a particular chromosome (the described NHP isapparently encoded on human chromosome 1, see GENBANK accession no.AL133380). These sequences identify actual, biologically verified, andtherefore relevant, exon splice junctions as opposed to those that mayhave been bioinformatically predicted from genomic sequence alone. Thesequences of the present invention are also useful as additional DNAmarkers for restriction fragment length polymorphism (RFLP) analysis,and in forensic biology.

[0008] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists, of NHP expression and/or NHP activity that utilize purifiedpreparations of the described NHPs and/or NHP product, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequence of a novel human ORFthat encodes the described novel human kinase protein. SEQ ID NO:3describes the NHP ORF and flanking regions.

5. DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHP described for the first time herein, is a novel proteinthat is expressed in, inter alia, human cell lines, and human fetalbrain, brain, pituitary, cerebellum, lymph node, trachea, kidney, liver,prostate, testis, thyroid, adrenal gland, pancreas, stomach, smallintestine, colon, skeletal muscle, heart, uterus, and fetal kidneycells. The described sequences were compiled from human genomic sequenceand cDNAs made from human brain, lymph node, liver, cerebellum, kidney,testis, and bone marrow mRNAs (Edge Biosystems, Gaithersburg, Md.,Clontech, Palo Alto, Calif.).

[0011] The present invention encompasses the nucleotides presented inthe Sequence Listing, host cells expressing such nucleotides, theexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described genes, including thespecifically described NHP, and the NHP products; (b) nucleotides thatencode one or more portions of the NHP that correspond to functionaldomains, and the polypeptide products specified by such nucleotidesequences, including but not limited to the novel regions of any activedomain(s); (c) isolated nucleotides that encode mutant versions,engineered or naturally occurring, of the described NHP in which all ora part of at least one domain is deleted or altered, and the polypeptideproducts specified by such nucleotide sequences, including but notlimited to soluble proteins and peptides in which all or a portion ofthe signal sequence is deleted; (d) nucleotides that encode chimericfusion proteins containing all or a portion of a coding region of a NHP,or one of its domains (e.g., a receptor/ligand binding domain, accessoryprotein/self-association domain, etc.) fused to another peptide orpolypeptide; or (e) therapeutic or diagnostic derivatives of thedescribed polynucleotides such as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing. As discussed above,the present invention includes: (a) the human DNA sequences presented inthe Sequence Listing (and vectors comprising the same) and additionallycontemplates any nucleotide sequence encoding a contiguous NHP openreading frame (ORF) that hybridizes to a complement of a DNA sequencepresented in the Sequence Listing under highly stringent conditions,e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3) and encodes a functionallyequivalent expression product. Additionally contemplated are anynucleotide sequences that hybridize to the complement of the DNAsequence that encode and express an amino acid sequence presented in theSequence Listing under moderately stringent conditions, e.g., washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet stillencode a functionally equivalent NHP product. Functional equivalents ofa NHP include naturally occurring NHPs present in other species andmutant NHPs whether naturally occurring or engineered (by site directedmutagenesis, gene shuffling, directed evolution as described in, forexample, U.S. Pat. Nos. 5,837,458 or 5,723,323 both of which are hereinincorporated by reference). The invention also includes degeneratenucleic acid variants of the disclosed NHP polynucleotide sequences.

[0012] Additionally contemplated are polynucleotides encoding NHP ORFs,or their functional equivalents, encoded by polynucleotide sequencesthat are about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NO:1 (as measured by BLAST sequence comparisonanalysis using, for example, the GCG sequence analysis package usingdefault parameters).

[0013] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP encoding polynucleotides. Such hybridizationconditions can be highly stringent or less highly stringent, asdescribed above. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80, or about 34 toabout 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0014] Alternatively, such NHP oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a micro array or high-throughput“chip” format). Additionally, a series of the described NHPoligonucleotide sequences, or the complements thereof, can be used torepresent all or a portion of the described NHP sequences. Anoligonucleotide or polynucleotide sequence first disclosed in at least aportion of one or more of the sequences of SEQ ID NOS: 1-3 can be usedas a hybridization probe in conjunction with a solid supportmatrix/substrate (resins, beads, membranes, plastics, polymers, metal ormetallized substrates, crystalline or polycrystalline substrates, etc.).Of particular note are spatially addressable arrays (i.e., gene chips,microtiter plates, etc.) of oligonucleotides and polynucleotides, orcorresponding oligopeptides and polypeptides, wherein at least one ofthe biopolymers present on the spatially addressable array comprises anoligonucleotide or polynucleotide sequence first disclosed in at leastone of the sequences of SEQ ID NOS: 1-3, or an amino acid sequenceencoded thereby. Methods for attaching biopolymers to, or synthesizingbiopolymers on, solid support matrices, and conducting binding studiesthereon are disclosed in, inter alia, U.S. Pat. Nos. 5,700,637,5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326,5,424,186, and 4,689,405 the disclosures of which are hereinincorporated by reference in their entirety.

[0015] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-3 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-3.

[0016] For example, a series of the described oligonucleotide sequences,or the complements thereof, can be used in chip format to represent allor a portion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

[0017] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-3 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

[0018] Probes consisting of sequences first disclosed in SEQ ID NOS:1-3can also be used in the identification, selection and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the drugs intended target.These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0019] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-3 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-3 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

[0020] Thus the sequences first disclosed in SEQ ID NOS:1-3 can be usedto identify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

[0021] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS: 1-3. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence. For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP gene antisense molecules, useful, for example,in NHP gene regulation and/or as antisense primers in amplificationreactions of NHP gene nucleic acid sequences. With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences can be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

[0022] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0023] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0024] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0025] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

[0026] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0027] Low stringency conditions are well-known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Spring Harbor Press, NY.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, NY.

[0028] Alternatively, suitably labeled NHP nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0029] For example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individual's genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

[0030] Further, a NHP gene homolog can be isolated from nucleic acidfrom an organism of interest by performing PCR using two degenerate or“wobble” oligonucleotide primer pools designed on the basis of aminoacid sequences within the NHP products disclosed herein. The templatefor the reaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue, such as prostate, rectum, colon, or adrenal gland,known or suspected to express an allele of a NHP gene.

[0031] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0032] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown to, or suspected of, expressing a NHP gene). A reversetranscription (RT) reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” using a standard terminaltransferase reaction, the hybrid may be digested with RNase H, andsecond strand synthesis may then be primed with a complementary primer.Thus, cDNA sequences upstream of the amplified fragment can be isolated.For a review of cloning strategies that can be used, see e.g., Sambrooket al., 1989, supra.

[0033] A cDNA encoding a mutant NHP sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known or suspected to be expressed in an individualputatively carrying a mutant NHP allele, and by extending the new strandwith reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal sequence. Using these two primers, the product isthen amplified via PCR, optionally cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well-known to thoseof skill in the art. By comparing the DNA sequence of the mutant NHPallele to that of a corresponding normal NHP allele, the mutation(s)responsible for the loss or alteration of function of the mutant NHPgene product can be ascertained.

[0034] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of or known to carry a mutant NHPallele (e.g., a person manifesting a NHP-associated phenotype such as,for example, immune disorders, obesity, high blood pressure, etc.), or acDNA library can be constructed using RNA from a tissue known, orsuspected, to express a mutant NHP allele. A normal NHP gene, or anysuitable fragment thereof, can then be labeled and used as a probe toidentify the corresponding mutant NHP allele in such libraries. Clonescontaining mutant NHP sequences can then be purified and subjected tosequence analysis according to methods well-known to those skilled inthe art.

[0035] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHP allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHP product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.)

[0036] Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to NHP are likely to cross-react with acorresponding mutant NHP expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well-known in theart.

[0037] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721 and 5,837,458 which are herein incorporated byreference in their entirety.

[0038] The invention also encompasses (a) DNA vectors that contain anyof the foregoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast a-mating factors.

[0039] Where, as in the present instance, some of the described NHPpeptides or polypeptides are thought to be cytoplasmic proteins,expression systems can be engineered that produce soluble derivatives ofa NHP (corresponding to a NHP extracellular and/or intracellulardomains, or truncated polypeptides lacking one or more hydrophobicdomains) and/or NHP fusion protein products (especially NHP-Ig fusionproteins, i.e., fusions of a NHP domain to an IgFc), NHP antibodies, andanti-idiotypic antibodies (including Fab fragments) that can be used intherapeutic applications. Preferably, the above expression systems areengineered to allow the desired peptide or polypeptide to be recoveredfrom the culture media.

[0040] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHP, as well as compounds or nucleotide constructs thatinhibit expression of a NHP sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHP (e.g., expression constructs in which NHP coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0041] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists ctn be useful for thedetection of mutant NHPs or inappropriately expressed NHPs for thediagnosis of disease. The NHP proteins or peptides, NHP fusion proteins,NHP nucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of NHP inthe body. The use of engineered host cells and/or animals can offer anadvantage in that such systems allow not only for the identification ofcompounds that bind to the endogenous receptor/ligand of a NHP, but canalso identify compounds that trigger NHP-mediated activities orpathways.

[0042] Finally, the NHP products can be used as therapeutics. Forexample, soluble derivatives such as NHP peptides/domains correspondingto the NHPs, NHP fusion protein products (especially NHP-Ig fusionproteins, i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHP, or a NHP-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the NHP could activateor effectively antagonize the endogenous NHP or a protein interactivetherewith. Nucleotide constructs encoding such NHP products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, well as antisense and ribozyme molecules can also be usedin “gene therapy” approaches for the modulation of NHP expression. Thus,the invention also encompasses pharmaceutical formulations and methodsfor treating biological disorders.

[0043] Various aspects of the invention are described in greater detailin the subsections below.

5.1 The NHP Sequences

[0044] The cDNA sequences and the corresponding deduced amino acidsequence of the described NHP are presented in the Sequence Listing. TheNHP nucleotide sequences were obtained from cDNAs obtained using probesand/or primers generated from human genomic sequence.

[0045] A number of polymorphisms that may occur in the described NHPwere identified including an A/G polymorphism at the nucleotide positionrepresented by, for example, position 2182 of SEQ ID NO: 1 (which canresult in a val or ile at the region corresponding to amino acid (aa)position 728 of, for example, SEQ ID NO:2), a G/T polymorphism atnucleotide position 2223 (which can result in an glu or asp at aaposition 741), a G/T polymorphism at nucleotide position 2350 (which canresult in a gly or cys at aa position 784), an A/C polymorphism atnucleotide position 2765 (which can result in an asp or ala at aaposition 922), a C/T polymorphism at nucleotide position 2768 (which canresult in a leu or pro at aa position 923), and an A/T polymorphism atnucleotide position 2773 (which can result in a ser or cys at aaposition 925).

[0046] Expression analysis has provided evidence that the described NHPsare predominantly expressed in CNS tissues, and that the NHP sharessignificant similarity with a variety of protein kinases. Given thephysiological importance of protein kinases, they have been subject tointense scrutiny as exemplified and discussed in U.S. Pat. Nos.5,756,289 and 5,817,479 herein incorporated by reference in theirentirety which additionally describe a variety of uses and applicationsfor the described NHP.

[0047] The described NHP is apparently encoded on human chromosome 1.

[0048] The described novel human polynucleotide sequences can be used,among other things, in the molecular mutagenesis/evolution of proteinsthat are at least partially encoded by the described novel sequencesusing, for example, polynucleotide shuffling or related methodologies.Such approaches are described in U.S. Pat. Nos. 5,830,721 and 5,837,458which are herein incorporated by reference in their entirety.

[0049] NHP gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate NHP transgenic animals.

[0050] Any technique known in the art may be used to introduce a NHPtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus-mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

[0051] The present invention provides for transgenic animals that carrythe NHP transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell-type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell-type ofinterest, and will be apparent to those of skill in the art.

[0052] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

[0053] The transgene can also be selectively introduced into aparticular cell-type, thus inactivating the endogenous NHP gene in onlythat cell-type, by following, for example, the teaching of Gu et al.,1994, Science, 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

[0054] Once transgenic animals have been generated, the expression ofthe recombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of MRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

5.2 NHP and NHP Polypeptides

[0055] NHP products, polypeptides, peptide fragments, mutated,truncated, or deleted forms of the NHPs, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as reagents in diagnosticassays, the identification of other cellular gene products related tothe NHP, as reagents in assays for screening for compounds that can beused as pharmaceutical reagents for the therapeutic treatment of mental,biological, or medical disorders and disease.

[0056] The Sequence Listing discloses the amino acid sequence encoded bythe described NHP-encoding polynucleotides. The NHP has an initiatormethionine in a DNA sequence context consistent with eucaryotictranslation initiation site and a signal-like sequence indicating thatthe NHP can be secreted or membrane associated.

[0057] The NHP amino acid sequence of the invention include the aminoacid sequence presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHP homologues from otherspecies are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al. eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0058] The invention also encompasses proteins that are functionallyequivalent to the NHP products encoded by the presently describednucleotide sequences as judged by any of a number of criteria,including, but not limited to, the ability to bind and modify a NHPsubstrate, or the ability to effect an identical or complementarydownstream pathway, or a change in cellular metabolism (e.g.,proteolytic activity, ion flux, tyrosine phosphorylation, etc.). Suchfunctionally equivalent NHP proteins include, but are not limited to,additions or substitutions of amino acid residues within the amino acidsequence encoded by the NHP nucleotide sequences described above, butwhich result in a silent change, thus producing a functionallyequivalent gene product. Amino acid substitutions can be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

[0059] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. Where the NHPpeptide or polypeptide can exist, or has been engineered to exist, as asoluble or secreted molecule, the soluble NHP peptide or polypeptide canbe recovered from the culture media. Such expression systems alsoencompass engineered host cells that express a NHP, or functionalequivalent, in situ. Purification or enrichment of a NHP from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well-known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of the NHP, but to assess biological activity, e.g., incertain drug screening assays.

[0060] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHPnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0061] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

[0062] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign polynucleotidesequences. The virus grows in Spodoptera frugiperda cells. A NHP codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of NHP coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted sequence is expressed (e.g., see Smith etal., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0063] In mammalian host cells, a number of viral-based expressionsystems can be utilized. In cases where an adenovirus is used as anexpression vector, the NHP nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene can then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHP product in infected hosts(e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHP nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHP gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHP coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, must be provided. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (See Bitter et al.,1987, Methods in Enzymol. 153:516-544).

[0064] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

[0065] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the NHP sequences described above can be engineered. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the NHP product. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds that affectthe endogenous activity of the NHP product.

[0066] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817)genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567;O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre etal., 1984, Gene 30:147).

[0067] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni²⁺-nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

[0068] Also encompassed by the present invention are fusion proteinsthat direct the NHP to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHPs to antibody moleculesor their Fab fragments could be used to target cells bearing aparticular epitope. Attaching the appropriate signal sequence to the NHPwould also transport the NHP to the desired location within the cell.Alternatively targeting of NHP or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes:A Practical Approach”, New,R.R.C., ed., Oxford University Press, New York and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of the NHP to the targetsite or desired organ, where they cross the cell membrane and/or thenucleus where the NHP can exert its functional activity. This goal maybe achieved by coupling of the NHP to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. applications Ser. No. 60/111,701 and 60/056,713,both of which are herein incorporated by reference, for examples of suchtransducing sequences) to facilitate passage across cellular membranesand can optionally be engineered to include nuclear localization.

5.3 Antibodies to NHP Products

[0069] Antibodies that specifically recognize one or more epitopes of aNHP, or epitopes of conserved variants of a NHP, or peptide fragments ofa NHP are also encompassed by the invention. Such antibodies include butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0070] The antibodies of the invention can be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHP expression product. Additionally, such antibodies can be usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHP-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHP activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

[0071] For the production of antibodies, various host animals may beimmunized by injection with the NHP, an NHP peptide (e.g., onecorresponding to a functional domain of an NHP), truncated NHPpolypeptides (NHP in which one or more domains have been deleted),functional equivalents of the NHP or mutated variant of the NHP. Suchhost animals may include but are not limited to pigs, rabbits, mice,goats, and rats, to name but a few. Various adjuvants may be used toincrease the immunological response, depending on the host species,including, but not limited to, Freund's adjuvant (complete andincomplete), mineral salts such as aluminum hydroxide or aluminumphosphate, chitosan, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Alternatively, the immune response could beenhanced by combination and or coupling with molecules such as keyholelimpet hemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, choleratoxin or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0072] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal, Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

[0073] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused (see U.S. Pat. Nos. 6,075,181 and 5,877,397 both of which areherein incorporated by reference in their entirety). A chimeric antibodyis a molecule in which different portions are derived from differentanimal species, such as those having a variable region derived from amurine mAb and a human immunoglobulin constant region. Such technologiesare described in U.S. Pat. Nos. 6,075,181 and 5,877,397 and theirrespective disclosures which are herein incorporated by reference intheir entirety. Also encompassed by the present invention is the use offully humanized monoclonal antibodies as described in U.S. Pat. No.6,150,584 and respective disclosures which are herein incorporated byreference in their entirety.

[0074] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0075] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: F(ab′)₂ fragments, which can be produced by pepsindigestion of the antibody molecule; and Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0076] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniqueswell-known to those skilled in the art. (See, e.g., Greenspan & Bona,1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to a NHP domain andcompetitively inhibit the binding of NHP to its cognate receptor/ligandcan be used to generate anti-idiotypes that “mimic” the NHP and,therefore, bind, activate, or neutralize a NHP, NHP receptor, or NHPligand. Such anti-idiotypic antibodies or Fab fragments of suchanti-idiotypes can be used in therapeutic regimens involving aNHP-mediated pathway.

[0077] Additionally given the high degree of relatedness of mammalianNHPs, the presently described knock-out mice (having never seen NHP, andthus never been tolerized to NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHP (i.e., NHP will be immunogenic in NHP knock-outanimals).

[0078] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 3 1 3111 DNA homo sapiens 1 atggctttgc ggggcgccgc gggagcgaccgacaccccgg tgtcctcggc cgggggagcc 60 cccggcggct cagcgtcctc gtcgtccacctcctcgggcg gctcggcctc ggcgggcgcg 120 gggctgtggg ccgcgctcta tgactacgaggctcgcggcg aggacgagct gagcctgcgg 180 cgcggccagc tggtggaggt gctgtcgcaggacgccgccg tgtcgggcga cgagggctgg 240 tgggcaggcc aggtgcagcg gcgcctcggcatcttccccg ccaactacgt ggctccctgc 300 cgcccggccg ccagccccgc gccgccgccctcgcggccca gctccccggt acacgtcgcc 360 ttcgagcggc tggagctgaa ggagctcatcggcgctgggg gcttcgggca ggtgtaccgc 420 gccacctggc agggccagga ggtggccgtgaaggcggcgc gccaggaccc ggagcaggac 480 gcggcggcgg ctgccgagag cgtgcggcgcgaggctcggc tcttcgccat gctgcggcac 540 cccaacatca tcgagctgcg cggcgtgtgcctgcagcagc cgcacctctg cctggtgctg 600 gagttcgccc gcggcggagc gctcaaccgagcgctggccg ctgccaacgc cgccccggac 660 ccgcgcgcgc ccggcccccg ccgcgcgcgccgcatccctc cgcacgtgct ggtcaactgg 720 gccgtgcaga tagcgcgggg catgctctacctgcatgagg aggccttcgt gcccatcctg 780 caccgggacc tcaagtccag caacattttgctacttgaga agatagaaca tgatgacatc 840 tgcaataaaa ctttgaagat tacagattttgggttggcga gggaatggca caggaccacc 900 aaaatgagca cagcaggcac ctatgcctggatggcccccg aagtgatcaa gtcttccttg 960 ttttctaagg gaagcgacat ctggagctatggagtgctgc tgtgggaact gctcaccgga 1020 gaagtcccct atcggggcat tgatggcctcgccgtggctt atggggtagc agtcaataaa 1080 ctcactttgc ccattccatc cacctgccctgagccgtttg ccaagctcat gaaagaatgc 1140 tggcaacaag accctcatat tcgtccatcgtttgccttaa ttctcgaaca gttgactgct 1200 attgaagggg cagtgatgac tgagatgcctcaagaatctt ttcattccat gcaagatgac 1260 tggaaactag aaattcaaca aatgtttgatgagttgagaa caaaggaaaa ggagctgcga 1320 tcccgggaag aggagctgac tcgggcggctctgcagcaga agtctcagga ggagctgcta 1380 aagcggcgtg agcagcagct ggcagagcgcgagatcgacg tgctggagcg ggaacttaac 1440 attctgatat tccagctaaa ccaggagaagcccaaggtaa agaagaggaa gggcaagttt 1500 aagagaagtc gtttaaagct caaagatggacatcgaatca gtttaccttc agatttccag 1560 cacaagataa ccgtgcaggc ctctcccaacttggacaaac ggcggagcct gaacagcagc 1620 agttccagtc ccccgagcag ccccacaatgatgccccgac tccgagccat acagttgact 1680 tcagatgaaa gcaataaaac ttggggaaggaacacagtct ttcgacaaga agaatttgag 1740 gatgtaaaaa ggaattttaa gaaaaaaggttgtacctggg gaccaaattc cattcaaatg 1800 aaagatagaa cagattgcaa agaaaggataagacctctct ccgatggcaa cagtccttgg 1860 tcaactatct taataaaaaa tcagaaaaccatgcccttgg cttcattgtt tgtggaccag 1920 ccagggtcct gtgaagagcc aaaactttcccctgatggat tagaacacag aaaaccaaaa 1980 caaataaaat tgcctagtca ggcctacattgatctacctc ttgggaaaga tgctcagaga 2040 gagaatcctg cagaagctga aagctgggaggaggcagcct ctgcgaatgc tgccacagtc 2100 tccattgaga tgactcctac gaatagtctgagtagatccc cccagagaaa gaaaacggag 2160 tcagctctgt atgggtgcac crtccttctggcatcggtgg ctctgggact ggacctcaga 2220 gakcttcata aagcacaggc tgctgaagaaccgttgccca aggaagagaa gaagaaacga 2280 gagggaatct tccagcgggc ttccaagtcccgcagaagyg ccagtcctcc cacaagcctg 2340 ccatccacck gtggggaggc cagcagcccaccctccctgc cactgtcaag tgccctgggc 2400 atcctctcca caccttcttt ctccacaaagtgcctgctgc agatggacag tgaagatcca 2460 ctggtggaca gtgcacctgt cacttgtgactctgagatgc tcactccgga tttttgtccc 2520 actgccccag gaagtggtcg tgagccagccctcatgccaa gacttgacac tgattgtagt 2580 gtatcaagaa acttgccgtc ttccttcctacagcagacat gtgggaatgt accttactgt 2640 gcttcttcaa aacatagacc rtcacatcacagacggacca tgtctgatgg aaatccgacc 2700 ccaactggtg caactattat ctcagccactggagcctctg cactgccact ctgcccctca 2760 cctgmtcytc acwgtcatct gccaagggaggtctcaccca agaagcacag cactgtccac 2820 atcgtgcctc agcgtcgccc tgcctccctgagaagccgct cagatctgcc tcaggcttac 2880 ccacagacag cagtgtctca gctggcacagactgcctgtg tagtgggtcg cccaggacca 2940 catcccaccc aattcctcgc tgccaaggagagaactaaat cccatgtgcc ttcattactg 3000 gatgctgacg tggaaggtca gagcagggactacactgtgc cactgtgcag aatgaggagc 3060 aaaaccagcc ggccatctat atatgaactggagaaagaat tcctgtctta a 3111 2 1036 PRT homo sapiens VARIANT(1)...(1036) Xaa = Any Amino Acid 2 Met Ala Leu Arg Gly Ala Ala Gly AlaThr Asp Thr Pro Val Ser Ser 1 5 10 15 Ala Gly Gly Ala Pro Gly Gly SerAla Ser Ser Ser Ser Thr Ser Ser 20 25 30 Gly Gly Ser Ala Ser Ala Gly AlaGly Leu Trp Ala Ala Leu Tyr Asp 35 40 45 Tyr Glu Ala Arg Gly Glu Asp GluLeu Ser Leu Arg Arg Gly Gln Leu 50 55 60 Val Glu Val Leu Ser Gln Asp AlaAla Val Ser Gly Asp Glu Gly Trp 65 70 75 80 Trp Ala Gly Gln Val Gln ArgArg Leu Gly Ile Phe Pro Ala Asn Tyr 85 90 95 Val Ala Pro Cys Arg Pro AlaAla Ser Pro Ala Pro Pro Pro Ser Arg 100 105 110 Pro Ser Ser Pro Val HisVal Ala Phe Glu Arg Leu Glu Leu Lys Glu 115 120 125 Leu Ile Gly Ala GlyGly Phe Gly Gln Val Tyr Arg Ala Thr Trp Gln 130 135 140 Gly Gln Glu ValAla Val Lys Ala Ala Arg Gln Asp Pro Glu Gln Asp 145 150 155 160 Ala AlaAla Ala Ala Glu Ser Val Arg Arg Glu Ala Arg Leu Phe Ala 165 170 175 MetLeu Arg His Pro Asn Ile Ile Glu Leu Arg Gly Val Cys Leu Gln 180 185 190Gln Pro His Leu Cys Leu Val Leu Glu Phe Ala Arg Gly Gly Ala Leu 195 200205 Asn Arg Ala Leu Ala Ala Ala Asn Ala Ala Pro Asp Pro Arg Ala Pro 210215 220 Gly Pro Arg Arg Ala Arg Arg Ile Pro Pro His Val Leu Val Asn Trp225 230 235 240 Ala Val Gln Ile Ala Arg Gly Met Leu Tyr Leu His Glu GluAla Phe 245 250 255 Val Pro Ile Leu His Arg Asp Leu Lys Ser Ser Asn IleLeu Leu Leu 260 265 270 Glu Lys Ile Glu His Asp Asp Ile Cys Asn Lys ThrLeu Lys Ile Thr 275 280 285 Asp Phe Gly Leu Ala Arg Glu Trp His Arg ThrThr Lys Met Ser Thr 290 295 300 Ala Gly Thr Tyr Ala Trp Met Ala Pro GluVal Ile Lys Ser Ser Leu 305 310 315 320 Phe Ser Lys Gly Ser Asp Ile TrpSer Tyr Gly Val Leu Leu Trp Glu 325 330 335 Leu Leu Thr Gly Glu Val ProTyr Arg Gly Ile Asp Gly Leu Ala Val 340 345 350 Ala Tyr Gly Val Ala ValAsn Lys Leu Thr Leu Pro Ile Pro Ser Thr 355 360 365 Cys Pro Glu Pro PheAla Lys Leu Met Lys Glu Cys Trp Gln Gln Asp 370 375 380 Pro His Ile ArgPro Ser Phe Ala Leu Ile Leu Glu Gln Leu Thr Ala 385 390 395 400 Ile GluGly Ala Val Met Thr Glu Met Pro Gln Glu Ser Phe His Ser 405 410 415 MetGln Asp Asp Trp Lys Leu Glu Ile Gln Gln Met Phe Asp Glu Leu 420 425 430Arg Thr Lys Glu Lys Glu Leu Arg Ser Arg Glu Glu Glu Leu Thr Arg 435 440445 Ala Ala Leu Gln Gln Lys Ser Gln Glu Glu Leu Leu Lys Arg Arg Glu 450455 460 Gln Gln Leu Ala Glu Arg Glu Ile Asp Val Leu Glu Arg Glu Leu Asn465 470 475 480 Ile Leu Ile Phe Gln Leu Asn Gln Glu Lys Pro Lys Val LysLys Arg 485 490 495 Lys Gly Lys Phe Lys Arg Ser Arg Leu Lys Leu Lys AspGly His Arg 500 505 510 Ile Ser Leu Pro Ser Asp Phe Gln His Lys Ile ThrVal Gln Ala Ser 515 520 525 Pro Asn Leu Asp Lys Arg Arg Ser Leu Asn SerSer Ser Ser Ser Pro 530 535 540 Pro Ser Ser Pro Thr Met Met Pro Arg LeuArg Ala Ile Gln Leu Thr 545 550 555 560 Ser Asp Glu Ser Asn Lys Thr TrpGly Arg Asn Thr Val Phe Arg Gln 565 570 575 Glu Glu Phe Glu Asp Val LysArg Asn Phe Lys Lys Lys Gly Cys Thr 580 585 590 Trp Gly Pro Asn Ser IleGln Met Lys Asp Arg Thr Asp Cys Lys Glu 595 600 605 Arg Ile Arg Pro LeuSer Asp Gly Asn Ser Pro Trp Ser Thr Ile Leu 610 615 620 Ile Lys Asn GlnLys Thr Met Pro Leu Ala Ser Leu Phe Val Asp Gln 625 630 635 640 Pro GlySer Cys Glu Glu Pro Lys Leu Ser Pro Asp Gly Leu Glu His 645 650 655 ArgLys Pro Lys Gln Ile Lys Leu Pro Ser Gln Ala Tyr Ile Asp Leu 660 665 670Pro Leu Gly Lys Asp Ala Gln Arg Glu Asn Pro Ala Glu Ala Glu Ser 675 680685 Trp Glu Glu Ala Ala Ser Ala Asn Ala Ala Thr Val Ser Ile Glu Met 690695 700 Thr Pro Thr Asn Ser Leu Ser Arg Ser Pro Gln Arg Lys Lys Thr Glu705 710 715 720 Ser Ala Leu Tyr Gly Cys Thr Val Leu Leu Ala Ser Val AlaLeu Gly 725 730 735 Leu Asp Leu Arg Glu Leu His Lys Ala Gln Ala Ala GluGlu Pro Leu 740 745 750 Pro Lys Glu Glu Lys Lys Lys Arg Glu Gly Ile PheGln Arg Ala Ser 755 760 765 Lys Ser Arg Arg Ser Ala Ser Pro Pro Thr SerLeu Pro Ser Thr Gly 770 775 780 Gly Glu Ala Ser Ser Pro Pro Ser Leu ProLeu Ser Ser Ala Leu Gly 785 790 795 800 Ile Leu Ser Thr Pro Ser Phe SerThr Lys Cys Leu Leu Gln Met Asp 805 810 815 Ser Glu Asp Pro Leu Val AspSer Ala Pro Val Thr Cys Asp Ser Glu 820 825 830 Met Leu Thr Pro Asp PheCys Pro Thr Ala Pro Gly Ser Gly Arg Glu 835 840 845 Pro Ala Leu Met ProArg Leu Asp Thr Asp Cys Ser Val Ser Arg Asn 850 855 860 Leu Pro Ser SerPhe Leu Gln Gln Thr Cys Gly Asn Val Pro Tyr Cys 865 870 875 880 Ala SerSer Lys His Arg Pro Ser His His Arg Arg Thr Met Ser Asp 885 890 895 GlyAsn Pro Thr Pro Thr Gly Ala Thr Ile Ile Ser Ala Thr Gly Ala 900 905 910Ser Ala Leu Pro Leu Cys Pro Ser Pro Asp Leu His Xaa His Leu Pro 915 920925 Arg Glu Val Ser Pro Lys Lys His Ser Thr Val His Ile Val Pro Gln 930935 940 Arg Arg Pro Ala Ser Leu Arg Ser Arg Ser Asp Leu Pro Gln Ala Tyr945 950 955 960 Pro Gln Thr Ala Val Ser Gln Leu Ala Gln Thr Ala Cys ValVal Gly 965 970 975 Arg Pro Gly Pro His Pro Thr Gln Phe Leu Ala Ala LysGlu Arg Thr 980 985 990 Lys Ser His Val Pro Ser Leu Leu Asp Ala Asp ValGlu Gly Gln Ser 995 1000 1005 Arg Asp Tyr Thr Val Pro Leu Cys Arg MetArg Ser Lys Thr Ser Arg 1010 1015 1020 Pro Ser Ile Tyr Glu Leu Glu LysGlu Phe Leu Ser 1025 1030 1035 3 3518 DNA homo sapiens 3 gcagcgccctgggcacgacc atggtgggac gtcgcccgcg gcttcgggga ccgctgcggc 60 agcagaggcggctggccagg aacgcgggcc gaggctggac cctttgggca gctagcccgt 120 gatctctgccgtcaccgatc gcgattccta ccccctcgcc ttcccccggc gccgacggcc 180 acaccgccggacgatgcgcg cccgcggccg cccgggaggc tgagcccagc ttcccgctcc 240 gccttccccgcgcagctgcc cccatggctt tgcggggcgc cgcgggagcg accgacaccc 300 cggtgtcctcggccggggga gcccccggcg gctcagcgtc ctcgtcgtcc acctcctcgg 360 gcggctcggcctcggcgggc gcggggctgt gggccgcgct ctatgactac gaggctcgcg 420 gcgaggacgagctgagcctg cggcgcggcc agctggtgga ggtgctgtcg caggacgccg 480 ccgtgtcgggcgacgagggc tggtgggcag gccaggtgca gcggcgcctc ggcatcttcc 540 ccgccaactacgtggctccc tgccgcccgg ccgccagccc cgcgccgccg ccctcgcggc 600 ccagctccccggtacacgtc gccttcgagc ggctggagct gaaggagctc atcggcgctg 660 ggggcttcgggcaggtgtac cgcgccacct ggcagggcca ggaggtggcc gtgaaggcgg 720 cgcgccaggacccggagcag gacgcggcgg cggctgccga gagcgtgcgg cgcgaggctc 780 ggctcttcgccatgctgcgg caccccaaca tcatcgagct gcgcggcgtg tgcctgcagc 840 agccgcacctctgcctggtg ctggagttcg cccgcggcgg agcgctcaac cgagcgctgg 900 ccgctgccaacgccgccccg gacccgcgcg cgcccggccc ccgccgcgcg cgccgcatcc 960 ctccgcacgtgctggtcaac tgggccgtgc agatagcgcg gggcatgctc tacctgcatg 1020 aggaggccttcgtgcccatc ctgcaccggg acctcaagtc cagcaacatt ttgctacttg 1080 agaagatagaacatgatgac atctgcaata aaactttgaa gattacagat tttgggttgg 1140 cgagggaatggcacaggacc accaaaatga gcacagcagg cacctatgcc tggatggccc 1200 ccgaagtgatcaagtcttcc ttgttttcta agggaagcga catctggagc tatggagtgc 1260 tgctgtgggaactgctcacc ggagaagtcc cctatcgggg cattgatggc ctcgccgtgg 1320 cttatggggtagcagtcaat aaactcactt tgcccattcc atccacctgc cctgagccgt 1380 ttgccaagctcatgaaagaa tgctggcaac aagaccctca tattcgtcca tcgtttgcct 1440 taattctcgaacagttgact gctattgaag gggcagtgat gactgagatg cctcaagaat 1500 cttttcattccatgcaagat gactggaaac tagaaattca acaaatgttt gatgagttga 1560 gaacaaaggaaaaggagctg cgatcccggg aagaggagct gactcgggcg gctctgcagc 1620 agaagtctcaggaggagctg ctaaagcggc gtgagcagca gctggcagag cgcgagatcg 1680 acgtgctggagcgggaactt aacattctga tattccagct aaaccaggag aagcccaagg 1740 taaagaagaggaagggcaag tttaagagaa gtcgtttaaa gctcaaagat ggacatcgaa 1800 tcagtttaccttcagatttc cagcacaaga taaccgtgca ggcctctccc aacttggaca 1860 aacggcggagcctgaacagc agcagttcca gtcccccgag cagccccaca atgatgcccc 1920 gactccgagccatacagttg acttcagatg aaagcaataa aacttgggga aggaacacag 1980 tctttcgacaagaagaattt gaggatgtaa aaaggaattt taagaaaaaa ggttgtacct 2040 ggggaccaaattccattcaa atgaaagata gaacagattg caaagaaagg ataagacctc 2100 tctccgatggcaacagtcct tggtcaacta tcttaataaa aaatcagaaa accatgccct 2160 tggcttcattgtttgtggac cagccagggt cctgtgaaga gccaaaactt tcccctgatg 2220 gattagaacacagaaaacca aaacaaataa aattgcctag tcaggcctac attgatctac 2280 ctcttgggaaagatgctcag agagagaatc ctgcagaagc tgaaagctgg gaggaggcag 2340 cctctgcgaatgctgccaca gtctccattg agatgactcc tacgaatagt ctgagtagat 2400 ccccccagagaaagaaaacg gagtcagctc tgtatgggtg caccrtcctt ctggcatcgg 2460 tggctctgggactggacctc agagakcttc ataaagcaca ggctgctgaa gaaccgttgc 2520 ccaaggaagagaagaagaaa cgagagggaa tcttccagcg ggcttccaag tcccgcagaa 2580 gygccagtcctcccacaagc ctgccatcca cckgtgggga ggccagcagc ccaccctccc 2640 tgccactgtcaagtgccctg ggcatcctct ccacaccttc tttctccaca aagtgcctgc 2700 tgcagatggacagtgaagat ccactggtgg acagtgcacc tgtcacttgt gactctgaga 2760 tgctcactccggatttttgt cccactgccc caggaagtgg tcgtgagcca gccctcatgc 2820 caagacttgacactgattgt agtgtatcaa gaaacttgcc gtcttccttc ctacagcaga 2880 catgtgggaatgtaccttac tgtgcttctt caaaacatag accrtcacat cacagacgga 2940 ccatgtctgatggaaatccg accccaactg gtgcaactat tatctcagcc actggagcct 3000 ctgcactgccactctgcccc tcacctgmtc ytcacwgtca tctgccaagg gaggtctcac 3060 ccaagaagcacagcactgtc cacatcgtgc ctcagcgtcg ccctgcctcc ctgagaagcc 3120 gctcagatctgcctcaggct tacccacaga cagcagtgtc tcagctggca cagactgcct 3180 gtgtagtgggtcgcccagga ccacatccca cccaattcct cgctgccaag gagagaacta 3240 aatcccatgtgccttcatta ctggatgctg acgtggaagg tcagagcagg gactacactg 3300 tgccactgtgcagaatgagg agcaaaacca gccggccatc tatatatgaa ctggagaaag 3360 aattcctgtcttaaactaag tgccttactg ttgtttaagc atttttttaa ggtgaacaaa 3420 tgaacacaatgtatctacct ttgaactgtt tcatgctgct gtgttttcaa aagctgtggc 3480 catgttcctaaattagtaag atatatccag cttctcaa 3518

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence that: (a) encodes the amino acid sequence shown inSEQ ID NO: 2; and (b) hybridizes under stringent conditions to thenucleotide sequence of SEQ ID NO: 1 or the complement thereof.
 2. Anisolated nucleic acid molecule comprising a nucleotide sequence encodingthe amino acid sequence shown in SEQ ID NO:2.